A comparison of callus induction in 4 Garcinia species

Background: This research is intended to determine suitable types and concentrations of plant growth regulators (PGRs) to induce callus on stem and leaf sections of 4 species of the genus Garcinia, namely, Garcinia mangostana, Garcinia schomburgkiana, Garcinia cowa, and Garcinia celebica. The base medium was MS medium containing 30 g l -1 sucrose, 0.5 g l-1 polyvinylpyrrolidone (PVP), and 7 g l-1 agar, and for the different treatments, PGRs were added to the medium as follows: thidiazuron (TDZ) at concentrations of 0, 0.1, 0.5, 1, and 2 mg l-1; 6-(3- hydroxybenzylamino) purine (meta-topolin) at concentrations of 0, 0.5, 2.5, and 5 mg l-1; 4-amino-3,5,6- trichloro-2-pyridinecarboxylic acid (picloram) at concentrations of 0, 0.5, 2.5, and 5 mg l-1; and 2,4- dichlorophenoxyacetic acid (2,4-D) at concentrations of 0, 0.5, 1, 2, and 4 mg l-1. The occurrence of callus was observed after 4 weeks. Results: A maximum of 100% and 93% of G. mangostana leaf explants formed callus in the 0.5 mg l-1 and 1 mg l-1 TDZ treatments, respectively, while 100% of G. schomburgkiana stem explants formed callus in the 1 mg l-1 TDZ treatment and 89% of G. schomburgkiana leaf explants formed callus in the 0.5 mg l-1 picloram treatment. The highest callus induction rate for G. cowa was 62% in the 1 mg l-1 TDZ treatment and for G. celebica was 56% in the 0.5 mg l-1•mT-1 treatment. Conclusions: For all 4 species, the greatest amount of large nodular callus was observed in the TDZ treatments. White, friable callus was observed on most of the 2,4-D and picloram treatment groups. Most meta-topolin treatments resulted in minimal callus formation.

Micropropagation of Calophyllum brasiliense (Cambess) from nodal segments / Micropropagação de Calophyllum brasiliense (Cambess) a partir de segmentos nodais

Abstract Micropropagation of Calophyllum brasiliense Cambess. (Clusiaceae) is a way to overcome difficulties in achieving large-scale plant production, given the recalcitrant nature of the seeds, irregular fructification and absence of natural vegetative propagation of the species. Cultures were established using nodal segments 2 cm in length, obtained from 1-2 year old seedlings, maintained in a greenhouse. Mercury chloride and Plant Preservative Mixture™ were used in the surface sterilizing stage, better results being achieved with Plant Preservative Mixture™ incorporation in culture medium, at any concentration. Polyvinylpyrrolidone, activated charcoal, cysteine, ascorbic acid or citric acid were added to the culture medium to avoid oxidation. After 30 days of culture, polyvinylpirrolidone and ascorbic acid gave better results, eliminating oxidation in most explants. For shoot multiplication, benzylaminopurine was used in concentrations of 4.4 and 8.8 µM in Woody Plant Medium, resulting in an average of 4.43 and 4.68 shoots per explant, respectively, after 90 days. Indole-3-butyric acid and α-naphthalene acetic acid were used to induce root formation, reaching a maximum rooting rate of 24% with 20µM α-naphthalene acetic acid. For acclimatization. the rooted plants were transferred to Plantmax® substrate and cultured in a greenhouse, reaching 79% of survival after 30 days and 60% after one year.

Resumo A micropropagação de Calophyllum brasiliense Cambess. (Clusiaceae) é uma maneira de superar dificuldades para sua produção em larga escala, devido à natureza recalcitrante das sementes, frutificação irregular e ausência de propagação vegetativa natural da espécie. Culturas foram estabelecidas utilizando segmentos nodais com 2 cm de comprimento, obtidos de plantas com 1 a 2 anos de idade, mantidas em casa de vegetação. Cloreto de mercúrio e Plant Preservative Mixture™ foram utilizados durante a etapa de desinfestação, com melhores resultados alcançados com a incorporação de Plant Preservative Mixture™ ao meio de cultura. Polivinilpirrolidona, carvão ativado, cisteína, ácido ascórbico ou ácido cítrico foram adicionados ao meio de cultura para evitar a oxidação dos explantes. Após 30 dias de cultivo, o uso de polivinilpirrolidona ou ácido ascórbico proporcionou melhores resultados, eliminando a oxidação na maioria dos explantes. Para multiplicação das brotações, benzilaminopurina foi usada em concentrações de 4.4 e 8.8 µM em meio WPM, resultando em uma média de 4.43 e 4.68 brotações por explante, respectivamente, após 90 dias. Ácido indol-3-butírico e ácido α-naftaleno acético foram usados para a indução de raízes, alcançando um enraizamento máximo de 24% com o uso de 20µM de ácido α-naftaleno acético. As plantas enraizadas foram transferidas para substrato Plantmax® e cultivadas em casa de vegetação, alcançando 79% de sobrevivência após 30 dias e 60% após um ano.

Effects of variations in culture media and hormonal treatments upon callus induction potential in endosperm explant of Barringtonia racemosa L

Objective: To induce callus from the medicinally valuable species, Barringtonia racemosa L. (B. racemosa) whereby the formation of callus is essential for micropropagation studies and in vitro plant secondary metabolites production. Methods: The callus induction potential in B. racemosa was assessed from endosperm explant cultured on different culture media and plant hormonal treatments. Lloyd and McCown's woody plant medium and Murashige and Skoog's medium were used in the study as culture media. On the other hand, various concentrations and combinations of 2,4-dichlorophenoxyacetic acid (1.0-2.0 mg/L) and kinetin (0.5-2.5 mg/L) had been incorporated in the culture media to exert the effects of auxin and cytokinin on callus induction. Results: From the present study, it was found that the profuse [(1.681 ± 0.770) g fresh weight, (0.239 ± 0.239) g dry weight] and friable callus formation was optimally produced with desirable morphology and considerable percentage of callus induction (56.70%) in endosperm explants cultured on 1.0 mg/L 2,4-dichlorophenoxyacetic acid and 1.5 mg/L kinetin in Murashige and Skoog's medium. Conclusions: A reliable protocol for inducing callus formation of profuse and friable morphology in endosperm explant of B. racemosa had therefore been successfully established.

In Vitro Regeneration from Shoot Tip and Nodal Explants of Simarouba Glauca DC, a Promising Biodiesel Tree.

An efficient regeneration protocol was developed from shoot tip and nodal explants of Simarouba glauca DC, a promising biodiesel plant. Nodal explants appeared to have better regeneration capacity than shoot tip explants (40%) in the tested media. The highest regeneration frequency (90%) and shoot number (7.00 ± 1.00 shoots per explants) were obtained in nodal explants in Murashige and Skoog’s (MS) medium supplemented with 6-benzylaminopurine (BAP) 4.43 μM and α-naphthalene acetic acid (NAA) 5.36 μM.Induced shoot buds were multiplied and elongated on the MS medium supplemented with BAP (4.44 μM), NAA (5.36 μM) and TDZ (Thidiazuron) 2.27 μM with 9.66±0.33 (mean length 5.35±0.32 cm) and 9.00±0.57 (mean length 4.51±0.15cm) shoots using nodal segments and shoot tip explants, respectively. Halfstrength woody plant medium (WPM) containing 2.46μM indole-3-butyric acid (IBA) produced the maximum number of roots (6.00±1.15). The rooted plantlets were hardened on MS basal liquid medium and subsequently in polycups containing sterile soil and vermiculite (1:1) and successfully established in pots.

Expressão transiente do gene uidA em explantes foliares de Eucalyptus saligna Sm. transformado via Agrobacterium tumefaciens / Transient expression of uidA gene in leaf explants of Eucalyptus saligna Sm. transformed via Agrobacterium tumefaciens

O objetivo desse trabalho foi avaliar a influência do pré-cultivo de explantes foliares e do meio de cultura na ressuspensão de Agrobacterium tumefaciens para infecção dos explantes. Os meios MS/2 (50% da concentração de sais) e MS N/2 (50% da concentração de NH4NO3 e KNO3) + PGR (1,0µM de TDZ (thidiazuron) + 0,1 µM de ANA (ácido naftalenoacético)) foram testados na ressuspensão da bactéria para infecção dos explantes. O pré-cultivo consistiu da manutenção dos explantes em meio de cultura para formação de calos (MS N/2 + PGR) durante um dia, sendo o tratamento sem pré-cultivo consistituído dos explantes após a excissão dos mesmos. Os explantes foram mantidos no escuro a 25 ± 2ºC mediante a utilização de plástico preto. O delineamento usado foi o inteiramente casualisado com 20 explantes. Os experimentos foram repetidos duas vezes. O meio MS/2 promoveu resultados superiores (22,4%) comparado ao meio MS N/2 + PGR (14,5%) para a percentagem de área com expressão do gene uidA. Aos 7 dias de cultivo em meio seletivo, a percentagem de área expressando o gene uidA foi 1,6 no MS/2 e 0% para o MS N/2 + PGR. O pré-cultivo produziu resultados superiores aos encontrados sem pré-cultivo, atingindo 31,4% de expressão transiente e no tratamento sem pré-cultivo 2,1%. Após 7 dias de cultivo em meio seletivo, a percentagem de área de expressão dos explantes do tratamento com pré-cultivo permaneceu 4,8% e 0% para o tratamento sem pré-cultivo. Os resultados indicam que o précultivo e ressuspensão da bactéria em meio MS/2 aumentaram a eficiência da expressão transiente do gene uidA em explantes foliares de E. saligna.

The aim of this research was to evaluate the effect of the pre-culture of leaf explants and the effect of the culture medium for the Agrobacterium tumefaciens resuspension to the explant infection. The media, MS/2 (half strength) and MS N/2 (10.3 mM NH4NO3 and 9.4 mM KNO3) + PGR (1.0 µM TDZ (thidiazuron) and 0.1 µM NAA (1-Naphthaleneacetic acid)) were tested for the bacteria resuspension. The pre-culture consisted of the maintenance of the explants on culture medium for callus formation (MS N/2+PGR) during one day and the treatment without pre-culture consisted of the use of the explants after the excision of the same ones. At the end of the co-culture, the MS/2 promoted results superiors to the MS N/2+PGR, and the area percentage that presented expression of the gene uidA was of 22.4% compared at 14.5%. To the 7 days of culture on a medium with kanamycin, the area percentage expressing the gene uidA was 1.6 in MS/2 and 0% for the MS N/2+PGR. At the end of the co-culture, the pre-culture produced results superiors to the found in the treatment without pre-culture, reaching 31.4% of expression and in the treatment without pre-culture 2.1%. After 7 days of culture on a medium with kanamycin, the area percentage of explant expression of the treatment with pre-culture stayed 4.8% and 0% for the treatment without pre-culture. The results indicate that the pre-culture and the bacteria resuspension in MS/2 increase the efficiency of the transient expression of the gene uidA in leaf explants of E. saligna.

Effects of donor plant age and explants on in vitro culture of Cedrela montanaMoritz ex Turcz

Para evaluar el potencial organogénico de Cedrela montanaMoritz ex Turcz, se colectaron explantes de árboles maduros (10-20 años) y juveniles (7-18 meses). Los primeros incluyeron yemas,hojas y nudos de brotes juveniles (ubicados hacia la parte basal deltronco) y rejuvenecidos (obtenidos a partir de estacas). Los segundoshojas, peciolos, nudos, entrenudos y nudos de brotes elongados invitro. Los nudos de árboles juveniles presentaron el mayor potencialorganogénico, ya que el 45,8% de los explantes presentaron elongaciónde yemas axilares y el 56,2% enraizamiento en medio sin reguladoresde crecimiento. El 51% de los brotes elongados formaron brotesadventicios con 0.5 μM NAA y 0.5 μM BA, el 30% con 0.5 μM NAAy 1 μM BA, y el 30% con 1 μM BA; y el 20% raíces con 0,5 μM NAA.La formación de raíces se vio estimulada con la adición de carbónactivado (5 gL-1) en el medio. El 80% de las plántulas regeneradas apartir de nudos y el 72,5% de las provenientes de brotes generadosin vitro se aclimataron exitosamente. Por el contrario, explantesde árboles maduros presentaron baja respuesta organogénica.Elongación de yemas axilares fue registrada solamente en 10.7% delos nudos de brotes juveniles y en 6.7% de aquellos provenientes debrotes rejuvenecidos. En conclusión, la edad de la planta donadoray el tipo de explante influyen sobre el potencial organogénico de C.montana. Este estudio contribuyó al conocimiento de la respuesta deesta especie bajo condiciones in vitro...

To evaluate the organogenic potential ofCedrela montana Moritz ex Turcz, explants from mature(10-20 year-old) and juvenile (7-18 month-old) treeswere collected. The first grouping included buds, leaves,and nodes derived from juvenile basal offshoots andrejuvenated shoots from cuttings. The second, includedleaves, petioles, nodes, internodes and nodes of in vitroelongated shoots. The highest organogenic potential wasobserved in nodes from juvenile trees: 45.8% of explantspresented axillary bud elongation, while 56.2% presentedrooting in a growth regulator free culture medium. Fiftyonepercent of elongated shoots produced adventitiousshoots with 0.5 μM NAA and 0.5 μM BA; 30% with0.5 μM NAA and 1 μM BA; and 30% with 1 μM BA.Twenty percent presented roots with 0.5 μM NAA. Rootformation was stimulated in a medium supplementedwith activated charcoal (5 gL-1). The acclimatizationof eighty percent of plantlets regenerated from nodes,and of 72.5% in vitro generated shoots was successful.On the contrary, mature trees material presented loworganogenic response. Axillary bud elongation wasrecorded just in 10.7% of explants from juvenile shootsand in 6.7% of explants from rejuvenated shoots. Inconclusion the age of donor plant and type of explantaffect the organogenic potential of C. montana. Thisstudy contributes to the understanding of this species’response under in vitro conditions...

Para avaliar o potencial organogênico da Cedrela montanaMoritz ex Turcz, explantes derivados de árvores adultas (10-20 anos)e jovens (7-18 meses) foram coletados. O primeiro incluiu brotos,folhas, e nós derivados de brotações jovens (localizado na direçãoda parte basal do tronco) e rejuvenescida (obtido a partir de estacas).O segundo incluía folhas, pecíolos, nós, entrenós e nós de brotosalongados in vitro. Maior potencial organogênico foi observado emnós de árvores jovens, em que o alongamento de brotos foi obtidoem 45,8% dos explantes e o enraizamento atingiu 56,2% em meiosem reguladores de crescimento. Brotos adventícios foram induzidasem 51% dos rebentos gerados in vitro com 0.5 μM NAA e 0.5 μM BA;30% de indução ocorreu com 0.5 μM NAA e 1 μM BA; 30% com 1μM BA. Raízes adventícias foram induzidas em 20% dos rebentoscom 0,5 μM NAA. Formação de raízes foi estimulada com carvão ativado(5 gL-1) no meio. 80% das plântulas regeneradas a partir de nós e 72,5%das plântulas a partir de brotações obtidas in vitro foram aclimatizadascom sucesso. Em contraste, explantes derivados de árvores adultasapresentou resposta organogênica baixo. Alongamento de brotos degemas axilares foi registrado somente em 10,7% dos nós de brotaçõesjovens e 6,7% das brotações ejuvenescidas. Em conclusão a idade daplanta doadora e o tipo de explante afeta o potencial de organogênesein vitro da C. montana. Este estudo contribuiu para o conhecimento daresposta desta espécie sob condições in vitro...